The present invention relates to obtaining a natural mixture of conjugated estrogens from the urine of pregnant mares.
Estrogens are used in medicine for hormone replacement therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of the disorders of the climacteric period which occur in women after natural or artificial menopause. In these case, natural mixtures of conjugated estrogens such as are found in the urine of pregnant mares have proved particularly effective and readily compatible.
The dissolved solids content in the urine of pregnant mares (=pregnant mares' urine, abbreviated hereafter as “PMU”) can naturally vary within wide ranges, and may generally lie in a range of 40 to 90 g dry matter per litre. In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2 to 5% by weight relative to the dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[(3-hydroxyphenyl)methyl]-2(3H)-furanone, known as HPMF. These may be present in free or conjugated form. The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulphuric acid semi-ester sodium salt (abbreviated hereafter as “sulphate salt”). The content of conjugated estrogens (calculated as estrogen sulphate salt) may be between 0.3 and 1% by weight, relative to the dry matter.
A suitable method is already known from WO 98/08526, with which a largely cresol- and HPMF-free mixture which is depleted in phenolic urine contents and contains the natural estrogen content of the PMU practically completely can be obtained in a solid-phase extraction on a semipolar, in particular non-ionic semipolar, polymeric adsorption resin. This extract is suitable as a starting material for the preparation of pharmaceuticals which contain the natural mixture of conjugated estrogens as active substance constituent.
In order to be able to be used as active substance constituent for pharmaceuticals, the natural mixture of conjugated estrogens which is obtained must however meet certain pharmaceutical specifications, e.g. the specification laid down in the USP (United States Pharmacopeia) or European Pharmacopeia. Also, the content of free estrogens relative to the dry matter should not exceed certain limit values. Normally the pharmaceutical specification requirements set are met by the mixtures of conjugated estrogens obtained from PMU in accordance with the method of WO 98/08526.
It has, however, turned out that the required pharmaceutical specifications, for example the above limit value of the maximum tolerable content of free estrogens, can often not be met when an “aged” urine, e.g. a PMU which has been stored or transported for relatively long periods and/or possibly at elevated temperatures, is used to obtain the natural mixture of conjugated estrogens. This ageing of the PMU should probably be ascribed to the fact that depending on the storage and/or transport conditions the content of conjugated estrogens can decrease over time or at elevated temperatures, while the content of undesirable free estrogens increases as a result. The original composition of the natural mixture of estrogens can thus be adversely changed in aged urine compared with fresh urine. For the natural mixture of conjugated estrogens obtained with the method of WO 98/08526, when the composition of an aged starting product is possibly changed there is often the risk that the extract obtained can no longer meet the required pharmaceutical specifications, in particular the maximum tolerable content of free estrogens. What is particularly disadvantageous about this is that batches obtained from aged PMU with the active-substance extract, which is valuable in itself, of the mixture of conjugated estrogens can then no longer be used for the production of a pharmaceutical preparation and therefore have to be discarded. The loss of a valuable natural pharmaceutical active substance, in view of the costly collection of PMU, which can only be carried out during certain periods of pregnancy, and the logistical boundary conditions which this involves in practice and from economic points of view, is highly unsatisfactory.